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Farzan 3/7/07 14:39 Page 639 Antiviral Therapy 12:639–650 Review Severe acute respiratory syndrome coronavirus entry as a target of antiviral therapies Jens H Kuhn1,2, Wenhui Li1, Sheli R Radoshitzky1, Hyeryun Choe 3 and Michael Farzan1* 1 Department of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Research Center, Southborough, MA, USA 2 Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany 3 Department of Pediatrics, Children’s Hospital, Harvard Medical School, Boston, MA, USA *Corresponding author: Tel: +1 508 624 8019; Fax: +1 508 786 3317; E-mail: [email protected] The identification in 2003 of a coronavirus as the aetiological agent of severe acute respiratory syndrome (SARS) intensified efforts to understand the biology of coronaviruses in general and SARS coronavirus (SARS-CoV) in particular. Rapid progress was made in describing the SARS-CoV genome, evolution and lifecycle. Identification of angiotensin-converting enzyme 2 (ACE2) as an obligate cellular receptor for SARS-CoV contributed to understanding of the SARS-CoV entry process, and helped to characterize two targets of antiviral therapeutics: the SARS-CoV spike protein and ACE2. Here we describe the role of these proteins in SARS-CoV replication and potential therapeutic strategies aimed at preventing entry of SARS-CoV into target cells. Introduction The viral order Nidovirales currently contains the three families Arteriviridae, Coronaviridae, and Roniviridae [1,2]. The viruses assigned to these families differ in morphology but have a similar genomic organization. The most prominent shared feature of the nidoviruses is transcription of a set of nested, subgenomic messenger RNAs [1,2]. Human pathogens are found exclusively among the two genera Coronavirus and Torovirus in the family Coronaviridae [2,3]. Coronavirions (genus Coronavirus) contain a single copy of a 28- to 31-kb-long, capped and polyadenylated, linear, single-stranded RNA of positive polarity, which is helically encapsidated by nucleocapsid (N) proteins and surrounded by membrane (M) proteins. The envelope contains protrusions (spike [S] proteins) that are major antigenic determinants of coronavirions, which are 100–120 nm in diameter [4]. Virions attach to cellsurface receptors via their spike proteins, which then mediate fusion with the host cell membrane. Genome replication occurs in the cytoplasm. After replication and maturation, coronaviruses bud from the endoplasmic reticulum–Golgi intermediate compartment [4]. Coronaviruses infect a wide spectrum of animal species, including many different mammals and birds in which they cause acute or chronic upper respiratory, © 2007 International Medical Press 1359-6535 gastrointestinal, hepatic, or central nervous system diseases. Classically, three distinct genetic and serological coronavirus groups are differentiated [4]. Human coronavirus 229E (HCoV-229E, group 1) and HCoVOC43 (group 2) are the aetiological agents of mild and usually self-resolving upper respiratory tract infections in otherwise healthy people or severe pneumonias in immunocompromised individuals [3]. Several pathogenic human coronaviruses (B814, HCoV-OC16, HCoVOC37 and HCoV-OC48) await characterization [3]. Emerging coronaviruses In recent years, three more pathogenic human coronaviruses have been identified. In 2003, a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), was identified as the aetiological agent of SARS [5–9], which had first emerged in November of 2002 in Guangdong Province, China. Infected people presented with an influenza-like disease beginning with dyspnoea, headaches, myalgia and pyrexia, followed by acute pneumonia and respiratory failure. SARS-CoV transmits via droplets, fomites and direct person-to-person contact, which is one of the reasons why SARS evolved into a pandemic. A total of 639 Farzan 3/7/07 14:39 Page 640 JH Kuhn et al. 8,096 cases and 774 deaths (lethality 9.6%) were recorded in Asia, Europe and North America before the outbreak was declared over after successful public health intervention in July of 2003 [10–14]. A second emergence of SARS was recorded during the winter of 2003–2004, again in Guangdong Province. During this limited outbreak, only four individuals developed the disease and all survived [15–17]. Since then, SARS-CoV seems to have disappeared because no other human cases, with the exception of laboratory infections [18,19], have been identified. The natural reservoir of SARS-CoV remains elusive. Animals kept and sold at a Guangdong market place are likely to have been the immediate origin of the virus found in humans; market Chinese ferret badgers (Melogale moschata), Himalayan palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) were later shown to harbour viruses similar to SARS-CoV [20]. Himalayan palm civets are of special interest because (i) SARS-CoV can persist in Himalayan palm civets for weeks [21], (ii) the infections of 2003–2004 were connected to restaurants that served Himalayan palm civet meat [16,17], and (iii) culling of Himalayan palm civets and their removal from markets dramatically reduced SARS case numbers. However, it is unlikely that the Himalayan palm civet or other marketplace animals serve as natural reservoirs for the virus. Only some marketplace Himalayan palm civets tested positive for SARS-CoV, whereas animals from farms and captured in the wild were found to be free of infection [22,23]. Also, molecular studies imply that the SARS-CoV genome was not at equilibrium in the Himalayan palm civet host [17,22]. In the past 2 years, numerous novel full and partial coronaviral genomes from all coronaviral groups have been obtained from bats [24–31], including SARS-CoV-like viruses from Chinese horseshoe bats (Rhinolophus spp.) [27,29–31]. The isolated viruses are genetically diverse and do not appear to cause disease in the bat hosts, suggesting that the precursor of SARS-CoV is harboured by a yet-unidentified bat species and was transmitted to Himalayan palm civets. In the laboratory, SARS-CoV isolates from the 2002–2003 epidemic infect cats [32], ferrets [32], hamsters [33] and cynomolgus and rhesus macaques [34,35], and can be adapted to infect mice [36–39]. Although these animals spontaneously clear infection [21,32] and transmission has only been observed among cats and ferrets [32], these observations suggest that SARS-CoV could readily jump species barriers. SARS-CoV found in humans and Himalayan palm civets and SARS-CoV-like genomes from bats are outliers of group 2 coronaviruses. For this reason the classical division of the genus Coronavirus has been questioned. Some researchers classify these novel viruses as a subgroup (group 2b), whereas others 640 consider them representatives of a novel group 4. Some of the recently discovered novel bat coronaviruses unrelated to SARS-CoV and SARS-CoV-like viruses have been assigned to a putative group 5 [24,25,40–43]. However, none of these new groups have yet been accepted by the International Committee on Taxonomy of Viruses. Two other human coronaviruses have been identified since the discovery of SARS-CoV. HCoV-NL63, a group 1 virus, has been implicated in conjunctivitis, croup and respiratory infections in children. Recent surveys suggest that this virus is distributed globally [44–52]. A third novel human coronavirus, HCoV-HKU1 (group 2), was isolated from a 71-year-old man with pneumonia [53], and has since been found in patients with respiratory diseases in many countries, including Australia [54], France [55] and the United States [56]. Coronavirus cell entry The cell, tissue and host tropism of coronaviruses, as well as their virulence, is mainly determined by the S proteins, which protrude from the virion as large and distinct club- or petal-shaped, 20–40-nm-long peplomers [57–62], and, in some cases, also by the haemagglutin–esterase (HE) proteins [4]. S proteins are class I fusion and type 1 transmembrane proteins [63,64]. They consist of a large ectodomain, a transmembrane anchor, and a short cytoplasmic tail. The ectodomain contains distinct N-terminal (S1) and Cterminal (S2) domains. S1 binds to the host cellsurface receptor via a distinct, independently folded receptor-binding domain (RBD) [65,66], in some cases inducing conformational changes that expose a fusion peptide embedded in S2. This in turn results in a reorganization of S2’s two heptad repeats (HR-1 and HR-2) into coiled coils [64,67], followed by fusion of the cellular plasma membrane and the viral envelope, thereby releasing the coronaviral nucleocapsid into the cytoplasm [64]. Prior to virion budding, S proteins are either cleaved between their S1 and S2 domains by a furin-like protease in the Sprotein-expressing and virus-producing cell before the S protein becomes incorporated into the budding virion (for instance, infectious bronchitis virus and murine hepatitis virus) [68,69], or remain uncleaved before incorporation into virions (for example, HCoV-229E, HCoV-NL63 and SARS-CoV) [70–72]. After receptor binding, SARS-CoV, but not HCoVNL63, is dependent on S protein cleavage by cathepsin L or S in an endosomal or lysosomal compartment or by exogenous proteases such as elastase, thermolysin or trypsin, which target a limited number of S protein cleavage sites [73–75]. Whereas S protein cleavage within virus-producing © 2007 International Medical Press Farzan 3/7/07 14:39 Page 641 SARS–ACE2 interaction cells is now understood to be a step in the activation of the membrane-fusion activity of the S2 domain, the function of lysosomal cathepsin cleavage remains to be determined. Several coronavirus cell-surface receptors are known (Table 1). The zinc metalloprotease aminopeptidase N (APN, CD13) is the receptor of the group 1 viruses canine coronavirus, feline infectious peritonitis virus, HCoV-229E, porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus [66,76–79], but is not a receptor for HCoV-NL63 [80]. The receptor of HCoV-OC43 remains elusive, but the receptors of other group 2 viruses have been identified. For instance, those of murine hepatitis virus and bovine coronavirus have been identified as members of the carcinoembryonic antigen-cell adhesion family and 9O-acetylated sialic acids, respectively [81,82]. Receptors for group 3 (avian coronaviruses) and putative group 5 viruses have not yet been identified. Identification of human ACE2 as a SARSCoV and HCoV-NL63 receptor Analyses of the SARS-CoV S protein revealed a relatively low (20–27%) amino acid sequence identity to the spikes of other coronaviruses [83,84]. Although SARSCoV spikes are not cleaved by a host cell protease into S1 and S2 subunits prior to budding, discrete SARSCoV S1 and S2 domains can be delineated by similarity to the subunits of cleaved S proteins [72,83,85]. Using the S1 domain, angiotensin-converting enzyme 2 (ACE2) was identified as the principal SARS-CoV receptor after immunoprecipitation from lysates of SARS-CoV-susceptible African green monkey kidney (Vero E6) cells and mass spectrometry analysis [86]. Following cellular overexpression, recombinant human ACE2 supported the formation of syncytia with Sprotein-expressing human embryonic kidney (HEK 293T) cells. A soluble form of ACE2, and anti-ACE2 Table 1. Viruses of the genus Coronavirus (Nidovirales: Coronaviridae) and their receptors, modified from [4] Classical subgroup Species Virus (abbreviation) Receptor 1 1 Canine coronavirus Feline coronavirus 1 1 1 1 Human coronavirus 229E ‘Human coronavirus NL63’ Porcine epidemic diarrhoea virus Transmissible gastroenteritis virus 2 Bovine coronavirus Canine coronavirus (CCoV) Feline coronavirus (FCoV) Feline infectious peritonitis virus (FIPV) Human coronavirus 229E (HCoV-229E) ‘Human coronavirus NL63 (HCoV-NL63)’ Porcine epidemic diarrhoea virus (PEDV) Porcine respiratory coronavirus (PRCoV) Transmissible gastroenteritis virus (TGEV) Bovine coronavirus 2 Human coronavirus OC43 Human coronavirus OC43 2 2 2 2 ‘Human coronavirus HKU1’ Human enteric coronavirus Murine hepatitis virus Porcine haemagglutinating encephalomyelitis virus Puffinosis coronavirus Rat coronavirus ‘Human coronavirus HKU-1 (HCoV-HKU1)’ Human enteric coronavirus (HECoV) Murine hepatitis virus (MHV) Porcine haemagglutinating encephalomyelitis virus (HEV) Puffinosis coronavirus (PCoV) Rat coronavirus (RtCoV)) Sialodacryoadenitis virus (SDAV) Severe acute respiratory syndrome coronavirus (SARS-CoV) Infectious bronchitis virus (IBV) Pheasant coronavirus (PhCoV) Turkey coronavirus (TCoV) ‘B814’ ‘Human coronavirus OC16’ ‘Human coronavirus OC37’ ‘Human coronavirus OC48’ Rabbit coronavirus (RbCoV) APN APN APN APN ACE2 APN APN APN N-acetyl-9-Oacetylneuraminic acid? N-acetyl-9-Oacetylneuraminic acid? ? ? CEACAMs ? 2 2 2 3 3 3 NC NC NC NC NC Severe acute respiratory syndrome coronavirus Infectious bronchitis virus Pheasant coronavirus Turkey coronavirus ? ? ? ? Rabbit coronavirus ? ? ? ACE2 N-Acetylneuraminic acid? ? ? ? ? ? ? ? Names not listed by the International Committee on Taxonomy of Viruses (ICTV) are marked by inverted commas. ACE2, angiotensin-converting enzyme 2; APN, aminopeptidase N (CD13); CEACAMs, carcinoembryonic antigen-related cell adhesion molecules; L-SIGN, liver/lymph-node-specific intercellular adhesion molecule-3grabbing integrin (CD209L, DC-SIGN2, DC-SIGNR); NC, not classified/tentative. Antiviral Therapy 12:4 Pt B 641 Farzan 3/7/07 14:39 Page 642 JH Kuhn et al. antisera, but not anti-ACE1 antisera, inhibited SARSCoV replication in Vero E6 cells. Finally, human ACE2 expression in otherwise SARS-CoV-resistant cells rendered them permissive to transduction with lentiviruses pseudotyped with S protein [71,86–88]. ACE2 was also identified independently after transduction of HeLa cells with a retrovirus cDNA library from Vero E6 cells, and flow-cytometry-based selection of cells that bound to a purified S protein fragment [89]. Additional surface proteins might contribute to efficient SARS-CoV cell entry. For instance, DC-SIGN (CD209), DC-SIGNR (L-SIGN, CD209L) and LSECtin enhanced infection of ACE2-expressing cells [90–93]. However, these molecules did not mediate infection in the absence of ACE2, suggesting that they are attachment factors rather than true alternative receptors. ACE2 is currently the only known SARS-CoV receptor and is likely to be central to replication of virus in infected animals. This latter assertion is supported by multiple lines of evidence. First, human ACE2 binds the SARS-CoV S1 domain specifically and with high affinity (1.7 nM) [94]. Second, all cell lines supporting efficient SARS-CoV infection express ACE2 [87,95,96]. Third, the efficiency of SARS-CoV infection in cats, chickens, dogs, ferrets, hamsters, Himalayan palm civets, humans, mice, minks, monkeys and rats, or their cells, directly correlates with the ability of the various ACE2 orthologues to promote SARS-CoV cell entry [97–102]. Fourth, SARS-CoV S protein and S protein mRNA were only detected in ACE2-expressing cells in the lungs and gastrointestinal tract of humans, which are major sites of SARS-CoV replication [103–107]. Fifth, the S protein ACE2-binding domain, its RBD, induces neutralizing antibodies in mice, and anti-S-protein antibodies that inhibit ACE2-Sprotein association protect hamsters and mice from SARS-CoV infection [108–115]. Finally, SARS-CoV cannot replicate in ACE2–/– mice, but transgenic mice expressing human ACE2 die from a SARS-like disease after infection [116,117]. ACE2 is a carboxy-metalloprotease that contains a single active site with a HEXXH zinc-binding motif. Notably, APN, the receptor of most group 1 coronaviruses, is also a zinc-binding metalloprotease [118–120]. The crystal structure of ACE2 demonstrates a claw-like structure. An open–closed conformational change of the claw is triggered upon ligand binding [121]. By cleaving a variety of regulatory peptides mainly involved in blood pressure homeostasis, including the potent vasoconstrictor angiotensin II, ACE2 appears to counterbalance the actions of the related molecule ACE, which cleaves the inactive peptide angiotensin I to the active angiotensin II [122], and which cannot function as a SARS-CoV receptor [87]. Furthermore, severe heart contractility defects were observed in mice after targeted disruption of 642 ACE2 [123,124]. The enzymatic activity of ACE2 does not contribute to its ability to function as a SARS-CoV receptor. Small molecule catalytic inhibitors did not disturb ACE2–S protein interaction, and even S protein bound to ACE2 did not alter the catalytic activity of the enzyme [97]. This observation is in accordance with results obtained for other coronaviruses. For instance, cell entry of the group 1 coronavirus porcine transmissible gastroenteritis virus is also independent of the proteolytic activity of its receptor, APN [125]. On the other hand, ACE2 proteolysis or S-protein-mediated ACE2 downregulation has been suggested to have a role in SARS pathogenesis [126]. ACE2 and the SARS-CoV and HCoV-NL63 RBDs A 193-amino-acid S1 fragment (residues 318–510) was identified as the SARS-CoV RBD (Figure 1). This protein fragment, which is distinct in structure and location from the RBDs of, for example, HCoV-299E and murine hepatitis virus, binds human ACE2 with higher affinity than full-length S1 [72,94,127]. The crystal structure of the SARS-CoV RBD revealed that it consists of two subdomains. The first subdomain, a five-stranded antiparallel β-sheet with three connecting α-helices, makes up the core of the RBD and is similar to analogous regions of other group 2 coronaviruses. The second subdomain is unique to SARS-CoV. This extended loop (residues 424–494), termed the receptorbinding motif (RBM), represents a gently curved surface (Figure 1) [128]. The base of this surface, a twostranded antiparallel β-sheet, cradles the N-terminal helix of ACE2 directly; one ridge of the surface contacts one ACE2 loop, whereas the other inserts between two different ACE2 loops of the N-terminal lobe, which is located away from the active site of the enzyme [128]. Curiously, HCoV-NL63 and SARS-CoV share the same human tissue tropism. Albeit with lower affinity, HCoV-NL63 also uses ACE2 as its main host cell receptor [80,129] despite the fact that HCoV-NL63 and SARS-CoV S proteins share no amino acid identity [130]. In fact, HCoV-NL63 S protein is related to that of HCoV-229E (56% sequence identity), which uses APN as a receptor. Mapping of the HCoV-NL63 RBD indicates that this RBD spans a broader and less discrete portion of the S1 domain [130]. ACE2 from other species The possibility of several different animal species as SARS-CoV hosts (among others, bats, Chinese ferret badgers, Himalayan palm civets and raccoon dogs) [20,24–31], the differences in susceptibility of different animals and cells to SARS-CoV infection [32–38,100], and the fact that certain SARS-CoV© 2007 International Medical Press Farzan 3/7/07 14:39 Page 643 SARS–ACE2 interaction Figure 1. The SARS-CoV RBD complexed to human ACE2 Conserved in group 2 SARS-CoV RBD RBM ACE2 The receptor-binding domain (RBD) has two distinctive domains, one conserved among group 2 coronaviruses, and the receptor-binding motif (RBM). The RBM is in direct contact with angiotensin-converting enzyme 2 (ACE2) and is apparently unique to severe acute respiratory syndrome coronavirus (SARS-CoV). Residues of the RBD and a critical RBM residue, threonine 487, are shown as dark spheres. This residue, a serine in most SARS-CoV isolates from Guangdong marketplace animals, is a threonine in all SARS-CoV isolates from the 2002–2003 SARS outbreak. This threonine confers high-affinity binding to human and palm civet ACE2. It is in direct contact with human ACE2 lysine residue 353 (shown as light spheres), which, in mutagenesis studies, has been found to be critical to the ACE2 role as a SARS-CoV receptor. neutralizing antibodies inhibit only certain viral strains [110] implied that there are significant ACE2 differences among species and the RBDs of different viral isolates. For instance, the ACE2 tissue distribution is comparable in humans, mice and rats. Nevertheless, murine ACE2 bound less efficiently to the SARS-CoV and HCoVNL63 S1 domains and supported S-protein-mediated pseudotype transduction less efficiently than human ACE2 [98,130]. Murine NIH 3T3 cells supported SARS-CoV replication one order of magnitude less efficiently than NIH 3T3 cells expressing human ACE2 [98]. Rat ACE2 did not support transduction with SARS-CoV S protein pseudotypes at all. However, the exchange of four amino acid residues of rat ACE2 for the equivalent residues found in human ACE2 yielded an efficient SARS-CoV receptor [98]: rat ACE2 residues 82–84 form a glycosylation site that is not present on Himalayan palm civet, mouse, and human Antiviral Therapy 12:4 Pt B ACE2, and residue 353 is a histidine in mouse and rat ACE2 but a lysine in Himalayan palm civet and human ACE2. Additional comparisons of ACE2 orthologues from other species such as cat, chicken, dog, hamster, macaques and mink further emphasized that the SARSCoV susceptibility of each species is directly correlated to individual crucial amino acid changes rather than the overall relatedness of all orthologues to each other [99,101]. Interestingly, ACE2 residue 353 is also lysine in cats, chicken, cows, dogs, macaques, mink and pigs, but some of these ACE2s (for example chicken) are inefficient SARS-CoV receptors nevertheless [99,101]. Therefore, post-translational modifications and additional particular residues of ACE2 might also influence SARS-CoV species sensitivity. S proteins from SARS-CoV isolates obtained in 2002–2003 (TOR2 isolate) and in 2003–2004 (GD03 isolate), and from SARS-CoV-like viruses from 643 Farzan 3/7/07 14:39 Page 644 JH Kuhn et al. Himalayan palm civets (SZ3 isolate) all bound Himalayan palm civet ACE2. However, GD03 and SZ3 proteins bound human ACE2 less efficiently than TOR2 S protein [97]. Again, exchanging specific residues of human ACE2 for those of Himalayan palm civet ACE2 enhanced binding of GD03 and SZ3 S proteins. Interestingly, amino acid sequence comparisons of the RBDs of SARS-CoV and SARS-CoV-like viruses revealed only few strain variations [110]. Swapping of RBD residues 479 and 487 of GD03 and SZ3 viruses for the equivalent residues of TOR2 virus increased the affinity for human ACE2 [97], but these exchanges had no effect on the affinity to mouse ACE2 [131]. Residue 479 is an asparagine or serine in all SARS-CoV strains isolated from humans, but a lysine in viruses isolated from Himalayan palm civets or raccoon dogs. This lysine is incompatible with human ACE2 binding, whereas palm civet ACE2 can efficiently bind S proteins expressing either asparagine or lysine [97]. S protein residue 487 is a threonine in all SARS-CoV isolates of the 2002–2003 SARS outbreak [132], but a serine in the isolates from the mild 2003–2004 outbreak and in almost all SARS-CoV-like viruses from palm civets and raccoon dogs. The change to serine resulted in an approximately 20-fold decrease in binding to human ACE2 [97]. An increase in binding resulted when a threonine was introduced into the SZ3 RBD. A threonine at position 487 also substantially increased association with Himalayan palm civet ACE2 [97]. Taken together, these data imply that only a few residues of the SARS-CoV RBD and the host species ACE2 determine the efficiency of S1–ACE2 interactions, that the observed lack of severity and transmission of the 2003–2004 SARS outbreak could be due to incomplete adaptation of GD03 viruses to human ACE2, and that Himalayan palm civets might be an important intermediate in the transfer of SARS-CoV to humans. SARS-CoV-like viruses from bats lack the RBM in the RBD-equivalent region, including most residues associated with ACE2 binding. This fact might explain the inability of these viruses to replicate in SARS-CoV-permissive cells [29,30,128], and suggests that bat SARS-CoV-like viruses do not use ACE2 as a host-cell receptor. These viruses might have acquired the RBM, perhaps through recombination with a HCoV-NL63-like virus in a Himalayan palm civet, before evolving into SARS-CoV. Host cell proteases involved in coronavirus cell entry The S proteins of several coronaviruses (for example, HCoV-OC43) are cleaved between their S1 and S2 domains by furin-like proteases in the producer cell prior to virion budding. In accordance with the 644 biosynthesis of class I fusion proteins of viruses belonging to other families (including retroviruses and orthomyxo- viruses), this cleavage is understood to be an activation step for the S2 domain to expose its fusion peptide, usually at the N-terminus. However, there are exceptions. For instance, filoviral spike proteins, which are also class I fusion proteins, remain fully functional even if the furin cleavage site is knocked out [133]; the S protein of the group 2 coronavirus murine hepatitis virus remains cleaved or uncleaved depending on cell type [134]; and various other coronaviruses (including all group 1 coronaviruses and SARS-CoV) do not possess a furin-like cleavage site at all. Additionally, the efficient cell entry of some coronaviruses (murine hepatitis virus type 2 and SARS-CoV), but not of others (HCoV-NL63), is dependent on proteolytic cleavage by lysosomal cysteine proteases (cathepsins L and S, but not B in the case of SARS-CoV) after the receptor-binding step [74,75,135] and is sensitive to lysosomotropic agents such as ammonium chloride or the proton ATPase inhibitor bafilomycin A. Exogenous trypsin or thermolysin can enhance SARS-CoV cell entry when added to virions already attached to their cell-surface receptors. This treatment also bypassed the entry inhibition induced by ammonium chloride and general or specific cathepsin inhibitors [73–75]. The true function of cathepsins in the SARS-CoV and murine hepatitis virus type 2 life cycles therefore remains to be determined. Therapeutic entry inhibition Potential targets for specific antivirals against SARS-CoV and HCoV-NL63 include the coronaviral spike proteins and their cognate receptor ACE2, and the coronaviral proteases, mRNA cap-1 methyl-transferases, NTPases/helicases and transcriptase–replicase complexes [136–138]. HIV-1 protease inhibitors, interferons and ribavirin were used empirically to treat SARS during the two recognized outbreaks, but the beneficial effect of these treatments, if any, is unclear [139], although interferons at least were effective in inhibiting SARS-CoV replication in tissue culture [136], and prophylactic treatment of SARS-CoV-infected cynomolgus macaques with pegylated interferon-α significantly reduced viral replication and excretion [140]. At the time of writing, there are still no specific treatments of proven value available to prevent or treat SARS or HCoV-NL63 infections. However, several promising vaccine candidates are now being evaluated. The rapid molecular characterization of the cell-entry determinants of both agents and the development of a mouse and non-human primate model for SARS suggest that the development and evaluation of an effective treatment regimen should be © 2007 International Medical Press Farzan 3/7/07 14:39 Page 645 SARS–ACE2 interaction possible in the near future. Any such treatment would have the advantage of neutralizing the viruses before they attach to a host cell or during the entry step itself, thereby possibly preventing the need for drug delivery into target cells. Several entry-specific approaches have been pursued. Sera of convalescent SARS patients contain high titres of SARS-CoV-neutralizing antibodies, and the administration of the sera themselves proved to be beneficial to acutely ill SARS patients [141,142]. These observations suggested that the virus itself could be neutralized by specific antibodies that prevent its binding to its receptor. Indeed, several studies have identified the SARS-CoV RBD to be a major immune response determinant that contains multiple important neutralizing epitopes [108,114,115,143–148], supporting the idea that virus neutralization occurs mainly by interruption of the S protein–ACE2 interaction. In contrast to many other viruses, the SARS-CoV RBD is exposed on, rather than hidden from, the surface of the S protein, perhaps reflecting the fact that SARS-CoV favours rapid transmission over immune escape. Consequently, neutralizing antibodies to the RBD are easily induced even if full-length S protein is used as an inoculum [109,147]. For instance, a recombinant human single-chain variable region fragment against the SARS-CoV (TOR2 isolate) S1 domain from non-immune human antibody libraries (80R scFV) inhibited the formation of syncytia between ACE2-expressing and S-protein-expressing HEK 293 T-cells, and efficiently neutralized the infection of Vero E6 cells with SARS-CoV (Urbani isolate of the 2002–2003 epidemic). 80R scFV competed with soluble ACE2 for association with the S1 domain, and bound S1 with high affinity (Kd=32.3 nM). A human immunoglobulin G1 (IgG1) form of 80R scFV (80R) bound S1 with higher affinity (Kd=1.59 nM) and neutralized SARS-CoV (Urbani isolate) with a 20-fold higher efficiency than 80R scFV (IC50=0.37 nM) [109]. 80R binds to a conformationally sensitive S protein fragment (residues 324–503) that is located within the RBD and precipitates a fusion protein consisting of the RBD and the Fc region of human IgG1 (RBD–Fc) as efficiently as protein A [110]. Just like other monoclonal antibodies that react with the RBD, 80R inhibited SARS-CoV replication in mice at doses usable in humans [108,110,144]. However, it is important to note that 80R does not neutralize all SARS-CoV strains. For instance, the GD03 isolate is totally resistant to 80R because of a D480G mutation that destroys the 80R epitope [110]. Another study employed the RBD as a subunit vaccine. RBD–Fc in combination with Freud’s complete adjuvant induced antibodies in intradermally immunized New Zealand Antiviral Therapy 12:4 Pt B white rabbits that completely inhibited infection of Vero E6 cells with 100× 50% tissue culture infective doses of SARS-CoV at a serum dilution of 1:10,240. These antibodies also inhibited the interaction of S1 with commercially available ACE2 [111]. The immunization of mice and rabbits with inactivated SARS-CoV induced antibodies that specifically recognized the RBD, inhibited the ACE2–RBD interaction and prevented cell transduction with S-protein-pseudotyped lentiviruses [149]. SARS-CoV-neutralizing antibodies recognizing the RBD could also be induced in BALB/c mice, rabbits and Chinese rhesus macaques by immunization with attenuated, modified vaccinia virus Ankara expressing the SARS-CoV Urbani isolate S protein [143]. However, one important safety concern in treating SARS patients with reconvalescent sera, monoclonal antibodies or antibody-inducing viral fragments is antibody-dependent enhancement (ADE) of infection (for a review see [150]). For instance, ADE has been observed in feline peritonitis virus entry into primary feline macrophages [151], and antibodies that neutralized entry of lentiviruses pseudotyped with S protein from human SARS-CoV isolates enhanced cell entry of lentiviruses pseudotyped with S protein derived from plam civet SARS-CoV isolates [152]. In addition to the use of antibodies, the RBD itself, or modified peptides thereof, could be used as a therapeutic because the RBD competitively inhibits virion binding to ACE2. In vitro, RBD–Fc inhibited transduction of ACE2-overexpressing target cells with lentiviruses pseudotyped with SARS-CoV Urbani isolate S protein with a lower 50% inhibitory concentration (IC50; 10 nM) than S1–Fc (50 nM) [94]. In a mouse model, RBD–Fc elicited high titres of long-lasting neutralizing antibodies and protected the animals from challenge with infectious virus [153]. Also, an RBD peptide, consisting of residues 471–503, specifically inhibited SARS-CoV cell entry in vitro [154]. Soluble, recombinant, enzymatically inactive ACE2, or modified polypeptides containing its SARS-CoVbinding domain, could also be used as a decoy to neutralize SARS-CoV in the bloodstream [95], because it would bind to the virions’ peplomers, thereby preventing binding to cell-surface ACE2. In vitro, enzymatically inactive, soluble ACE2–Fc inhibited lentiviral pseudotype transduction more efficiently than S1–Fc, at an IC50 of 2 nM [71,97]. Furthermore, an ACE2-derived peptide, consisting of residues 22–44 and 351–357 linked by a glycine, exhibited anti-SARS-CoV activity with an IC50 of 100 nM [155]. Antibodies to ACE2 inhibited SARS-CoV replication in Vero E6 cells with an IC50 of 1.5 µg/ml [86]. On the basis of the available crystal structure of ACE2 complexed with the SARS-CoV RBD [128] it should 645 Farzan 3/7/07 14:39 Page 646 JH Kuhn et al. also be possible to develop specific proteinaceous inhibitors of ACE2 that do not contain RBD fragments or even small molecules that could interrupt ACE2–RBD interaction. At least one such inhibitor, a molecule that prevents both binding of SARS-CoV to ACE2 and ACE2 enzymatic activity, has been identified [156]. Other small molecules, VE607 and Emodin, inhibited SARS-CoV cell entry or pseudotype transduction in a dose-dependent manner, but it is not yet clear whether the molecules act on S protein or on ACE2 [157,158]. Because SARS-CoV binds to ACE2 at a location distinct from its active site [128] it should be possible to find inhibitors that only prevent SARS-CoV binding, but do not disturb the, probably important, physiological function of ACE2. Interestingly, both SARS-CoV and HCoV-NL63 use ACE2 as their receptors and both bind to overlapping but not identical sites away from ACE2’s catalytic centre [80,86,89,130]. This fact could permit the development of an inhibitor that targets both viruses at the same time. After binding to ACE2, SARS-CoV and HCoV-NL63 S proteins initiate fusion with the host cell membrane following a mechanism similar to that induced by other class I fusion proteins such as those of filoviruses, orthomyxoviruses and retroviruses. Experiments suggest that receptor binding occurs at neutral pH and that no viral proteins other than the spike proteins are required for this step and subsequent fusion [72,86]. The conformational changes of S2’s two heptad repeat regions HR-1 and HR-2 are critical for efficient fusion. Therefore, inhibiting the HR1–HR-2 interaction might prove to be another antiviral strategy. Indeed, synthetic peptides derived from the SARS-CoV HR-2 region that bound efficiently to HR-1 peptides interrupted the conformational changes and inhibited SARS-CoV infection, albeit with relatively low efficacy [159–162]. It could also be possible to develop SARS-CoVspecific antivirals based on host-cell protease inhibitors since efficient SARS-CoV entry is dependent on the activity of the lysosomal cysteine protease cathepsin L or S [74,75]. For instance, cell transduction with lentiviral particles pseudotyped with SARS-CoV S protein was inhibited in vitro by leupeptin (a serine and cysteine protease inhibitor), E64c (a cysteine protease inhibitor) and Z-lll-FMK (a cathepsin B and L inhibitor) at 95% inhibitory concentrations of 15.2, 8.2 and 3.5 µM, respectively [74,163]. Conclusions It remains unclear whether a SARS-CoV-like virus will re-emerge to threaten public health, as SARSCoV did in the winter of 2002–2003. 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Proteolysis of SARSassociated coronavirus spike glycoprotein. Adv Exp Med Biol 2006; 581:235–240. Accepted for publication 9 February 2007 650 © 2007 International Medical Press